Preparation of Tara Sample DNA From Iron-Chloride Precipitates
Locate samples and record inventory number and number of filters (or portions) used.
Turn the filters precipitate-side out with bleached forceps that have been rinsed with water and use aluminum foil squares as work surface, discarding after each filter.
Remove filter from 50cc tube and refold so that precipitate side comes in contact with resuspension buffer Return filter to tube using forceps and sterile applicator sticks.
Keep filters dark and refrigerated until ready to suspend.
Prepare 1x or 2x resuspension buffer.
Prepare 20mL 1x or 10mL 2x per filter from 20L of seawater.
Add resuspension buffer to each filter Parafilm the tubes and wrap all of them in aluminum foil.
Put foil pack of tubes on rotator, in cold room, using rubber bands to secure, and rotate slowly overnight.
To recover resuspended viruses, remove the liquid at the bottom using a 5mL pipet and transfer to a fresh 15mL or 50mL sterile, labeled tube.
Using bleached and rinsed forceps or sterile applicator sticks, pull edge of filter up and over lip of tube and secure with the lid.
Centrifuge 1000rpm, 5min, 18°C to recover liquid left on filter.
Remove liquid and add more buffer if filter still has a lot of precipitate clinging to it.
Rotate for several more hours.
Repeat removal of liquid.
Dilute stock DNase 1:100 in 10x DNase buffer (concentration = 400 U/mL).
Add 1/10th volume of diluted DNase to each sample Parafilm tubes, wrap them in aluminum foil and attach to tube rotator with rubber bands.
Incubate by rotating slowly at room temperature for 2 hours.
Inactivate DNase by adding EDTA and EGTA to 0.1M final concentration each.
Mix by inverting the tube several times.
Add the DNase treated and inactivated sample to the top reservoir of Amicon Ultra 100kDA centrifugal concentrators.
Centrifuge the concentrators at 1000 g for 5 minute intervals at 18°C until samples are at less than 2mL each.
Put 1mL of resin on one Wizard Prep column.
Thoroughly resuspend resin by shaking vigorously.
Mix 1mL per 0.5-1.0mL of DNA.
Add each 1mL of resin to a 3mL luer lock syringe attached to a Wizard column.
Push through into a 5mL snap-cap tube.
Save this tube until DNA quantification.
Remove syringe from column Remove plunger.
Reattach syringe barrel to column.
Wash columns with 2mL 80% isopropanol pushing through with the syringe barrel into a waste container.
Put columns into 1.5mL centrifuge tube and centrifuge at 10,000 g for 2.5 minutes to remove residual alcohol.
Discard tube.
Place column into a fresh 1.5mL centrifuge tube and pipet on 50-100µL of TE (0.02µm filtered and heated to 80°C).
Vortex gently (1400rpm) and let sit 1 minute.
Centrifuge at 10,000 g for 1 minute.
Transfer extracted DNA to Lo-bind DNA 0.5mL tubes.
Repeat elution above (steps 37-40) one more time.
Use 1-2µL of the first elution and 4-5µL of the second elution for the Pico Green assay to quantify the extracted DNAQuant-iT dsDNA Pico Green assay kit (Invitrogen).
Use the Excel spreadsheet to calculate the ng/µL DNA for each sample.
Store in -80°C ultra-low freezer.
Label tubes with date, station, depth and concentration and store in -80°C ultra-low freezer
